The long-range goal of the proposed study is to better understand the role of Heat Shock Factor (Hsf) in protein folding homeostasis. The role of Hsf as a transcriptional regulator that is induced by protein folding stress has been studied for decades. More recently, studies of aging model organisms have revealed that Hsf function declines with age, thus limiting healthspan and lifespan. However, we still have a rudimentary understanding of how connections between Hsf and specific protein folding pathways are severed in old cells. This knowledge is critical for guiding ongoing therapeutic efforts to ameliorate the negative effects of aging on protein folding homeostasis. Hsf is conserved from yeast to humans. Yeast has powerful tools for studying gene expression and undergoes a form of cell aging known as replicative cell senescence that is under the control of conserved aging mechanisms. However, Hsf has been difficult to study in yeast because it is an essential protein even in the absence of protein folding stress (i.e. ?hsf cells are dead). Existing methods to conditionally inactivate Hsf are slow, thus raising concerns that they induce secondary gene expression effects unrelated to Hsf's essential function. Moreover, it is not known whether Hsf activity declines during yeast cell replicative senescence. We have developed a chemical genetics approach that rapidly and potently inhibits Hsf by mislocalizing it from the nucleus, where it normally resides in yeast, to the cytoplasm. Our plan is to combine this tool with genomic analysis of nascent transcripts to define the immediate effects of Hsf inactivation on gene expression and use this knowledge to engineer viable ?hsf cells (Aim 1). We have also established that Hsf activity declines as a function of replicative yeast cell age. Surprisingly, w found that an alternative transcriptional pathway maintains protein folding in aged cell by turning on many but not all Hsf gene targets. Our data also suggest that one of the protein folding pathways that is not maintained in aged cells is the Hsp90 protein folding pathway, which comprises many strict Hsf gene targets. We will test if the Hsf connection with Hsp90 is severed in old cells using a variety of single-cell reporters of Hsp90 function (Aim 2). The proposed studies will define how protein folding pathways are severed from Hsf transcriptional control in old cells and enable targeted therapies of Hsf effectors that extend healthspan and lifespan.